Compositions and methods for enhancing oil content in plants

ABSTRACT

The present invention provides transgenic plants with altered oil content. The invention further provides nucleic acid sequences and methods for generating such plants.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/937,026, filed Oct. 8, 2010, which is a National Phase of PCT PatentApplication No. PCT/IL2009/000394, filed Apr. 7, 2009, which claims thebenefit of priority of U.S. Provisional Application Nos. 61/159,092filed Mar. 11, 2009 and 61/043,747 filed Apr. 10, 2008. The contents ofthe above applications are all incorporated by reference as if fully setforth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 59845SequenceListing.txt, created on Jul. 3,2014, comprising 38,182 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to microRNAs and related nucleic acid sequencesassociated with enhanced oil content in plants, as well as to methodsfor generating transgenic plants with enhanced oil content.

BACKGROUND OF THE INVENTION

Biofuel is defined as solid, liquid or gaseous fuel derived fromrelatively recently dead biological material (and is therebydistinguished from fossil fuels, which are derived from long deadbiological material). Theoretically, biofuels can be produced from any(biological) carbon source; although, the most common sources arephotosynthetic plants. Various plants and plant-derived materials areused for biofuel manufacturing. Agrofuels are biofuels which areproduced from specific crops, rather than from waste processes.

There are two common strategies of producing liquid and gaseousagrofuels. One is to grow crops high in sugar (sugar cane, sugar beet,and sweet sorghum) or starch (corn/maize), and then use yeastfermentation to produce ethyl alcohol (ethanol). The second is to growplants that contain high amounts of vegetable oil, such as oil palm,soybean, algae, jatropha, or pongamia pinnata. When these oils areheated, their viscosity is reduced, and they can be burned directly in adiesel engine, or they can be chemically processed to produce fuels suchas biodiesel. Wood and its byproducts can also be converted intobiofuels such as methanol or ethanol fuel.

Algae can be used as a source for several types of biofuels with themost studied one being biodiesel. Algae are fast-growing organisms withnaturally high oil content that can be extracted year-round. Algae canbe grown in non-arable areas and thus do not compete with food crops onland. Algae provide the most promising long-term solution forsustainable biofuels production with only a minimal cropping arearequired to supplement a large fuel supply. Interest in algae isrecently growing as a potential feedstock for biodiesel production withthe emerging concern about global warming that is associated with theincreased usage of fossil fuels.

Algal biotechnology has made considerable advances in recent yearsincluding development of methods for genetic transformation andsequencing the genomes of several algal species and significant effortshave been made over the last 15 years to increase the oil yield inalgae. Oil content in algae is known to be influenced by severalenvironmental and nutritional factors such as nitrogen starvation andstrong light. Although much has been learned about genetic control ofoil synthesis in algae over that time, researchers have still not beenable to significantly increase the oil yield of algae by geneticengineering.

MicroRNAs are 17-24 nucleotide long, endogenous RNAs that regulate geneexpression in plants and animals. MicroRNAs are processed from stem-loopregions of long primary transcripts and are loaded into silencingcomplexes, where in plants they generally direct cleavage ofcomplementary mRNAs. MicroRNAs play crucial roles at each major stage ofplant development, typically at the cores of gene regulatory networks,usually targeting genes that are themselves regulators thus affectingthe abundance or stability of numerous genes at once.

Recently, microRNAs have been discovered in the unicellular model algaChlamydomonas reinhardtii and preliminary evidence indicates they playcrucial roles in algal development (Molnar et al., 2007, Nature447:1126-1130).

So far, plant microRNAs that target genes involved in abiotic and bioticstress responses, hormone signaling and metabolism and the microRNAmachinery itself have been identified (Jones-Rhoades et al., 2006, AnnuRev Plant Biol 57:19-53).

A commonly-used approach in identifying the function of novel genes isthrough a loss-of-function mutant screening. In many cases, functionalredundancy exists between genes that are members of the same family.When this happens, a mutation in one gene member might have a reduced oreven non-existing phenotype and the mutant lines might not be identifiedin the screening.

Using microRNAs, multiple members of the same gene family can besilenced simultaneously, giving rise to much more intense phenotypes.This approach is also superior to RNA interference (RNAi) techniqueswhere typically 100-800 bp fragments of the gene of interest form afold-back structure when expressed. These long fold-back RNAs form manydifferent small RNAs and prediction of small RNA targets other then theperfectly complementary intended targets are therefore very hard.MicroRNAs in contrast, are produced from precursors, which are normallyprocessed such that preferentially one single stable small RNA isgenerated, thus significantly minimizing the “off-target” effect.

A second approach of functional screening is through overexpression ofgenes of interest and testing for their phenotypes. In many cases,attempting to overexpress a gene which is under microRNA regulationresults in no significant increase in the gene's transcript. This can beovercome either by expressing a microRNA-resistant version of the geneor by downregulating the microRNA itself.

Attempts to increase oil yield in algae through standard molecularbiology techniques was so far only marginally successful. In recentyears, major effort was focused on studying the Acetyl-CoA carboxylase(ACCase) enzyme, which is considered to regulate a key step in fattyacid biosynthesis. While efforts focused on genetic manipulation toincrease the activity of ACCase have been going on for at least 15years, the research has not yet reached the stage of actually being ableto substantially increase the net oil yield from algae.

There is an unmet need to efficiently introduce significant changes inplants traits such as oil content.

SUMMARY OF THE INVENTION

The present invention provides transgenic plants with altered oilcontent. The invention further provides nucleic acid sequences andmethods for generating such plants.

According to one aspect, the present invention provides a transgenicplant transformed with an isolated nucleic acid selected from the groupconsisting of a microRNA, a microRNA precursor, a microRNA target, amicroRNA-resistant target, a fragment thereof, a nucleic acid sequencecapable of hybridizing thereto under stringent conditions, a nucleicacid sequence having at least 80% identity thereto, and combinationsthereof, wherein modulated expression of the isolated nucleic acidsequence results in altered oil content in said transgenic plantcompared to a corresponding wild type plant.

According to one embodiment, the nucleic acid sequence is selected fromthe group consisting of SEQ ID NOS: 14, 17, 35, 48, 51, 66, 78, 1-13,15-16, 18-34, 36-47, 49-50, 52-65, 67-75, 80, 84 and 85, a complementarysequence thereof, a fragment thereof, a nucleic acid sequence capable ofhybridizing thereto under stringent conditions, and sequences having atleast 80% identity thereto, and wherein the alteration is increased oilcontent.

According to some embodiments the nucleic acid sequence is selected fromthe group consisting of SEQ ID NOS: 4, 6, 8, 12-15, 17, 20, 24-26,29-31, 34-36, 41, 43, 44, 46-49, 51, 52, 55-57, 60-62, 65-67, 80, 84 and85, a fragment thereof, a nucleic acid sequence capable of hybridizingthereto under stringent conditions and sequences having at least 80%identity thereto, wherein the modulated expression is upregulation ofsaid nucleic acid sequence. According to some embodiments thetransformation comprises introduction of said nucleic acid sequence intosaid plant.

Also provided is a transgenic plant transformed with an isolated nucleicacid selected from the group consisting of SEQ ID NOS: 14, 17, 35, 48,51, 66 and 78, a fragment thereof, a nucleic acid sequence capable ofhybridizing thereto under stringent conditions and sequences having atleast 80% identity thereto, wherein the transformation comprisesintroduction of the nucleic acid sequence into the plant, and whereinthe transformation results in enhanced oil content in said transgenicplant compared to a corresponding wild type plant.

According to some embodiments the transformation results indown-regulated expression of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOS: 1, 5, 9, 16, 21, 23, 27, 28, 32, 37-39,40, 42, 45, 50, 53, 54, 58, 59, 63 and 68-75, a fragment thereof, anucleic acid sequence capable of hybridizing thereto under stringentconditions and sequences having at least 80% identity thereto, and thealteration is increased oil content.

According to some embodiments, the transformation comprises introductionof a microRNA-resistant target of a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 1, 5, 9, 16, 21, 23, 27, 28, 32,37-39, 40, 42, 45, 50, 53, 54, 58, 59, 63 and 68-75, a fragment thereof,a nucleic acid sequence capable of hybridizing thereto under stringentconditions and sequences having at least 80% identity thereto into saidplant. According to some embodiments, the sequence of the introducedmicroRNA-resistant target is selected from the group consisting of SEQID NOS: 86-88, a fragment thereof, a nucleic acid sequence capable ofhybridizing thereto under stringent conditions and sequences having atleast 80% identity thereto.

According to another aspect, the present invention provides a vectorcomprising a nucleic acid sequence associated with oil content of aplant, wherein said nucleic acid sequence is selected from the groupconsisting of a microRNA, a microRNA precursor, a microRNA target, amicroRNA-resistant target, a fragment thereof, a nucleic acid sequencecapable of hybridizing thereto and a nucleic acid sequence having atleast 80% identity thereto.

According to some embodiments the nucleic acid sequence is selected fromthe group consisting of SEQ ID NOS: 4, 6, 8, 12-15, 17, 20, 24-26,29-31, 34-36, 41, 43, 44, 46-49, 51, 52, 55-57, 60-62, 65-67, 80, 84 and85, a fragment thereof, a nucleic acid sequence capable of hybridizingthereto and a nucleic acid sequence having at least 80% identitythereto.

According to other embodiments the sequence is a microRNA-resistanttarget of a sequence selected from the group consisting of SEQ ID NOS:1, 5, 9, 16, 21, 23, 27, 28, 32, 37-39, 40, 42, 45, 50, 53, 54, 58, 59,63 and 68-75, a fragment thereof, a nucleic acid sequence capable ofhybridizing thereto and a nucleic acid sequence having at least 80%identity thereto. According to further embodiments the sequence of themicroRNA-resistant target is selected from the group consisting of SEQID NOS: 86-88, a fragment thereof, a nucleic acid sequence capable ofhybridizing thereto and a nucleic acid sequence having at least 80%identity thereto.

According to another aspect, the present invention provides a method foraltering the oil content in a plant, comprising modulating, in saidplant, the expression of a nucleic acid sequence selected from the groupconsisting of a microRNA, a microRNA precursor, a microRNA target, amicroRNA-resistant target, a fragment thereof, a nucleic acid sequencecapable of hybridizing thereto under stringent conditions, a nucleicacid sequence having at least 80% identity thereto and combinationsthereof.

According to some embodiments the nucleic acid sequence is selected fromthe group consisting of SEQ ID NOS: 14, 17, 35, 48, 51, 66, 78, 1-13,15-16, 18-34, 36-47, 49-50, 52-65, 67-75, 80, 84 and 85, a complementarysequence thereof, a fragment thereof, a nucleic acid sequence capable ofhybridizing thereto under stringent conditions and a nucleic acidsequence having at least 80% identity thereto, and said alteration isincreased oil content.

According to some embodiments the nucleic acid sequence is selected fromthe group consisting of SEQ ID NOS: 4, 6, 8, 12-15, 17, 20, 24-26,29-31, 34-36, 41, 43, 44, 46-49, 51, 52, 55-57, 60-62, 65-67, 80, 84 and85, a fragment thereof, a nucleic acid sequence capable of hybridizingthereto under stringent conditions and a nucleic acid sequence having atleast 80% identity thereto, and the modulation comprises overexpressingsaid nucleic acid sequence by its introduction it into said plant.

Also provided in accordance with the invention is a method for enhancingthe oil content in a plant, comprising introducing into said plant anucleic acid sequence selected from the group consisting of SEQ ID NOS:SEQ ID NOS: 14, 17, 35, 48, 51, 66 and 78, a fragment thereof, a nucleicacid sequence capable of hybridizing thereto under stringent conditionsand a nucleic acid sequence having at least 80% identity thereto,wherein expression of the introduced nucleic acids enhances the oilcontent in said plant.

According to some embodiments the modulation results in downregulatedexpression of a nucleic acid sequence is selected from the groupconsisting of SEQ ID NOS: SEQ ID NOS: 1, 5, 9, 16, 21, 23, 27, 28, 32,37-39, 40, 42, 45, 50, 53, 54, 58, 59, 63 and 68-75, a fragment thereof,a nucleic acid sequence capable of hybridizing thereto under stringentconditions and a nucleic acid sequence having at least 80% identitythereto, and the alteration is increased oil content.

According to some embodiments the method comprises introducing amicroRNA-resistant target of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOS: SEQ ID NOS: 1, 5, 9, 16, 21, 23, 27, 28,32, 37-39, 40, 42, 45, 50, 53, 54, 58, 59, 63 and 68-75, a fragmentthereof, a nucleic acid sequence capable of hybridizing thereto understringent conditions and a nucleic acid sequence having at least 80%identity thereto, and the alteration is increased oil content. Accordingto further embodiments the sequence of the microRNA-resistant target isselected from the group consisting of SEQ ID NOS: 86-88, a fragmentthereof, a nucleic acid sequence capable of hybridizing thereto understringent conditions and a nucleic acid sequence having at least 80%identity thereto.

According to some embodiments of the invention, the expression of theintroduced nucleic acid is driven by a promoter expressible in a plantcell selected from the group consisting of a constitutive, inducible,and tissue-specific promoter, operably linked to the nucleic acid.

According to some embodiments, the transgenic plant of the invention isselected from the group consisting of Corn, Rapeseed/Canola, Soybean,Sugarcane, sugar beet, Barley, Wheat, Jatropha, Castor bean, ChineseTallow, Palm, Camelina, and Mustard. According to some embodiments theplant is algae. According to some embodiments the algae selected fromthe group consisting of Neochloris oleoabundans, Scenedesmus dimorphus,Euglena gracilis, Phaeodactylum tricornutum, Pleurochrysis carterae,Tetraselmis chui, Tetraselmis suecica, Isochrysis galbana,Nannochloropsis salina, Nannochloropsis oculata, Botryococcus braunii,Dunaliella tertiolecta, Nannochloris sp., Spirulina sp., Chlorella sp.,Crypthecodinium cohnii, Cylindrotheca sp., Dunaliella primolecta,Monallanthus salina, Nitzschia sp., Schizochytrium sp. and Tetraselmissueica. According to some embodiments the algae is selected from thegroup consisting of Chlamydomonas reinhardtii, Chlorella vulgaris andPhaeodactylum tricornutum.

According to another aspect, the invention provides a seed obtained froma transgenic plant of the invention. The invention further provides aseed obtained by use of a vector of the invention, and a seed obtainedby use of a method of the invention.

These and other embodiments of the present invention will becomeapparent in conjunction with the figures, description and claims thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows oil content in C. reinhardtii in response to the nitrogenstarvation and strong light inductions. The Y-axis represents thepercent of oil content as compared to control and the X-axis representsthe time of cell harvesting for oil content analysis. The white barsrepresent the strong-light induction, and the light gray bars representthe nitrogen starvation induction.

FIGS. 2A-2D show oil content in transformed lines of C. reinhardtiioverexpressing different microRNAs. The Y-axis represents the oilcontent in the transformed lines relative to the oil content in the wildtype line grown under the same conditions. The X-axis present groups ofresults for several clones of each line, each clone designated with anumber. The black and white bars represent experiments preformed in 50ml tubes containing 10 ml of TAP growth medium, and thediagonally-striped bars represent experiments preformed in 250 ml flaskscontaining 50 ml of TAP growth medium.

FIG. 2A shows results for line transformed with a sequence consisting ofthe mature microRNA sequence of CRMIR5 inserted in the cre-mir1162backbone (CRMIR5 replacing mature cre-mR1162) (SEQ ID NO: 78).

FIG. 2B shows results for the line transformed with cre-mir1151b (SEQ IDNO: 48).

FIG. 2C shows results for the line transformed with cre-mir1157 (SEQ IDNO: 51).

FIG. 2D shows results for the line transformed with CRMIR121 (SEQ ID NO:66).

FIG. 3 shows the microRNA array results in response to growth of C.reinhardtii in nitrate-limiting conditions (Bristol medium withoutNaNO₃) or nitrate-sufficient conditions (standard Bristol medium). TheY-axis shows the mean normalized expression level of four experimentsconducted in nitrate-limiting conditions, and the X-axis shows the meannormalized expression level of four controls grown in nitrate-sufficientconditions. The middle diagonal line represents the expected expressionfor non-differentially expressed miRNAs (same expression level innitrate-sufficient and nitrate-limiting conditions) and the otherdiagonal lines represent fold 1.5 factor lines. Significantlydifferentially expressed miRs include cre-miR1142 (SEQ ID NO: 71),cre-miR1167 (SEQ ID NO: 21), CRMIR129, (SEQ ID NO: 37), CRMIR136 (SEQ IDNO: 72) and CRMIR137 (SEQ ID NO: 73). All of the significantlydifferentially expressed miRs were found to be downregulated in responseto nitrate-limiting conditions.

FIGS. 4A-4E are boxplot presentations comparing distributions of theexpression values (Y axis) of miRs with differential expression innitrate-limiting conditions (the left box) vs. nitrate-sufficientconditions (the right box). The line in the box indicates the medianexpression value, the box top and bottom boundaries indicate the 75thand 25th percentiles respectively, and the horizontal lines and crosses(outliers) show the full range of signals in each group.

FIG. 4A presents the differential expression of CRMIR137 (SEQ ID NO: 73)which is downregulated in nitrate-limiting conditions relative tonitrate-sufficient conditions with a p-value of 0.02 and a fold changeof 1.9.

FIG. 4B presents the differential expression of cre-miR1142 (SEQ ID NO:71) which is downregulated in nitrate-limiting conditions relative tonitrate-sufficient conditions with a p-value of 0.04 and a fold changeof 1.6.

FIG. 4C presents the differential expression of cre-miR1167 (SEQ ID NO:21) which is downregulated in nitrate-limiting conditions relative tonitrate-sufficient conditions with a p-value of 0.03 and a fold changeof 1.6.

FIG. 4D presents the differential expression of CRMIR129 (SEQ ID NO: 37)which is downregulated in nitrate-limiting conditions relative tonitrate-sufficient conditions with a p-value of 0.02 and a fold changeof 1.5.

FIG. 4E presents the differential expression of CRMIR136 (SEQ ID NO: 72)which is downregulated in nitrate-limiting conditions relative tonitrate-sufficient conditions with a p-value of 0.02 and a fold changeof 1.5.

FIG. 5 is a boxplot presentation comparing distributions of theexpression values (Y axis) of the adenylate cyclase-encoding gene ADCY33(SEQ ID NO: 80) in nitrate-limiting conditions (the right box) vs.nitrate-sufficient conditions (the left box). The line in the boxindicates the median expression value, the box top and bottom boundariesindicate the 75th and 25th percentiles respectively, and the horizontallines and crosses (outliers) show the full range of signals in eachgroup.

DETAILED DESCRIPTION OF THE INVENTION

According to some aspects of the present invention, oil production wasinduced in algae by nitrogen starvation and strong light, and isolatednucleic acid sequences, including microRNAs and microRNA precursors,associated with oil synthesis were identified. Additionally, lines of C.reinhardtii transformed to overexpress microRNA precursors associatedwith oil synthesis were found to have increased oil content relative tocontrols.

Before the present compositions and methods are disclosed and described,it is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 areexplicitly contemplated.

1. General Definitions

About

As used herein, the term “about” refers to +/−10%.

Algae

Algae include organisms that belong to the phyla Chlorophyta (greenalgae), Eustigmatophyceae (eustigmatophytes), Phaeophyceae (brownalgae), Bacillariophyta (diatoms), Xanthophyceae (yellow-green algae),Haptophyceae (prymnesiophytes), Chrysophyceae (golden algae), Rhodophyta(red algae) and Cyanobacteria, (blue-green algae). Specific examples ofalgae include, but are not limited to, Neochloris oleoabundans,Scenedesmus dimorphus, Euglena gracilis, Phaeodactylum tricornutum,Pleurochrysis carterae, Tetraselmis chui, Tetraselmis suecica,Isochrysis galbana, Nannochloropsis salina, Botryococcus braunii,Dunaliella tertiolecta, Nannochloris sp., Spirulina sp., Chlorella sp.,Crypthecodinium cohnii, Cylindrotheca sp., Dunaliella primolecta,Monallanthus salina, Nitzschia sp., Schizochytrium sp. and Tetraselmissueica.

Auxotrophy

As used herein, auxotrophy is the inability of an organism to synthesizea particular organic compound required for its growth.

Binding Site

As used herein, the term “binding site” may comprise a target nucleicacid or a portion thereof. The binding site has a perfect ornear-perfect complementarity to one of the miRNA sequences as listed inSanger database, version 10.1 or the database of Molnar et al, 2007.

Biodiesel

As used herein, the term “Biodiesel” refers to a non-petroleum-baseddiesel fuel consisting of long-chain alkyl (methyl, propyl or ethyl)esters. Biodiesel is made by chemically-reacting lipids, typicallyvegetable oil or animal fat, and alcohol. It can be used (alone, orblended with conventional petrodiesel) in unmodified diesel-engines.

Complement

“Complement” or “complementary” as used herein to refer to a nucleicacid may mean Watson-Crick or Hoogsteen base pairing between nucleotidesor nucleotide analogs of nucleic acid molecules. A full complement orfully complementary means 100% complementary base pairing betweennucleotides or nucleotide analogs of nucleic acid molecules.

Differential Expression

“Differential expression” means qualitative or quantitative differencesin the temporal and/or spatial gene expression patterns within and amongcells and tissue. Thus, a differentially expressed gene mayqualitatively have its expression altered, including an activation orinactivation, in, e.g., standard conditions versus environmental ornutritional stress. Genes may be turned on or turned off in a particularstate, relative to another state thus permitting comparison of two ormore states. A qualitatively regulated gene may exhibit an expressionpattern within a state or cell type which may be detectable by standardtechniques. Some genes may be expressed in one state or cell type, butnot in both. Alternatively, the difference in expression may bequantitative, e.g., in that expression is modulated, up-regulated,resulting in an increased amount of transcript, or down-regulated,resulting in a decreased amount of transcript. The degree to whichexpression differs needs only be large enough to quantify via standardcharacterization techniques such as expression arrays, quantitativereverse transcriptase PCR, Northern blot analysis, real-time PCR, insitu hybridization and RNAse protection.

Expression Profile

The term “expression profile” is used broadly to include a genomicexpression profile, e.g., an expression profile of microRNAs. Profilesmay be generated by any convenient means for determining a level of anucleic acid sequence e.g. quantitative hybridization of microRNA,labeled microRNA, amplified microRNA, cDNA, etc., quantitative PCR,ELISA for quantification, and the like, and allow the analysis ofdifferential gene expression between two samples. Samples are collectedby any convenient method, as known in the art. Nucleic acid sequences ofinterest are nucleic acid sequences that are found to be predictive,including the nucleic acid sequences provided below, where theexpression profile may include expression data for 5, 10, 20, 25, 50 ormore of, including all of the listed nucleic acid sequences. Accordingto some embodiments, the term “expression profile” means measuring theabundance of the nucleic acid sequences in the measured samples.

Feedstock

Feedstocks refer to the crops or products that can be used as orconverted into biofuels and bioenergy. Each feedstock has advantages anddisadvantages in terms of how much usable material they yield, wherethey can grow and how energy and water-intensive they are. As usedherein, second generation feedstocks refers broadly to crops that havehigh potential yields of biofuels, but that are not widely cultivated,or not cultivated as an energy crop.

Gene

“Gene” as used herein may be a natural (e.g., genomic) or synthetic genecomprising transcriptional and/or translational regulatory sequencesand/or a coding region and/or non-translated sequences (e.g., introns,5′- and 3′-untranslated sequences). The coding region of a gene may be anucleotide sequence coding for an amino acid sequence or a functionalRNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA. Agene may also be an mRNA or cDNA corresponding to the coding regions(e.g., exons) optionally comprising 5′- or 3′-untranslated sequenceslinked thereto. A gene may also be an amplified nucleic acid moleculeproduced in vitro comprising all or a part of the coding region and/or5′- or 3′-untranslated sequences linked thereto.

Identity

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences mean that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA sequences, thymine (T) and uracil (U) may be consideredequivalent. Identity may be performed manually or by using a computersequence algorithm such as BLAST or BLAST 2.0.

MicroRNA-resistant Target

As used herein, “microRNA-resistant target” is a microRNA targetmodified such that the microRNA cannot bind and microRNA mediated targetregulation is therefore prevented.

Modulated (or: Modified) Expression

As used herein, modulated expression in reference to a nucleic acidsequence indicates that the pattern of expression in, e.g., a transgenicplant or plant tissue, is different from the expression pattern in awild-type plant or a reference plant of the same species. The pattern ofexpression may also be compared with a reference expression pattern in awild-type plant of the same species. For example, the nucleic acid isexpressed in a cell or tissue type other than a cell or tissue type inwhich the sequence is expressed in the wild-type plant, or by expressionat a time other than at the time the sequence is expressed in thewild-type plant, or by a response to different inducible agents, such ashormones or environmental signals, or at different expression levels(either higher or lower) compared with those found in a wild-type plant.The term also refers to modulated expression patterns that are producedby lowering the levels of expression to below the detection level orcompletely abolishing expression. The resulting expression pattern canbe transient or stable, constitutive or inducible. The modulatedexpression may be of either or both of an endogenous nucleic acid, or anexogenous nucleic acid introduced to the plant.

Overexpression

The term “overexpression” as used herein refers to a greater expressionlevel of a gene in a plant, plant cell or plant tissue, compared toexpression in a wild-type plant, cell or tissue, at any developmental ortemporal stage for the gene. Overexpression can occur when, for example,the genes encoding one or more genes or transcription factors are underthe control of a strong expression signal. Overexpression may occurthroughout a plant or in specific tissues of the plant, depending on thepromoter used. Overexpression results in a greater than normalproduction, or “overproduction” of a gene product in a plant, cell ortissue.

Plant

Plants include anything under the kingdom “Viridiplantae”, e.g. Corn,Rapeseed/Canola, Soybean, Sugarcane, sugar beet, Barley, Wheat,Jatropha, Castor bean, Chinese Tallow, Palm, and Mustard. Plants alsoinclude Green algae, and as used herein, plants further include algaethat belong to the phyla Eustigmatophyceae (eustigmatophytes),Phaeophyceae (brown algae), Bacillariophyta (diatoms), Xanthophyceae(yellow-green algae), Haptophyceae (prymnesiophytes), Chrysophyceae(golden algae), Rhodophyta (red algae) and Cyanobacteria, (blue-greenalgae).

Probe

“Probe” as used herein means an oligonucleotide capable of binding to atarget nucleic acid of complementary sequence through one or more typesof chemical bonds, usually through complementary base pairing, usuallythrough hydrogen bond formation. Probes may bind target sequenceslacking complete complementarity with the probe sequence depending uponthe stringency of the hybridization conditions. There may be any numberof base pair mismatches which will interfere with hybridization betweenthe target sequence and the single stranded nucleic acids describedherein. However, if the number of mutations is so great that nohybridization can occur even under the least stringent of hybridizationconditions, the sequence is not a complementary target sequence. A probemay be single stranded or partially single and partially doublestranded. The strandedness of the probe is dictated by the structure,composition, and properties of the target sequence. Probes may bedirectly labeled or indirectly labeled such as with biotin to which astreptavidin complex may later bind.

Promoter

A sequence of nucleotides from which transcription may be initiated ofDNA operably linked downstream (i.e. in the 3′ direction on the sensestrand of double-stranded DNA). A promoter “drives” transcription of anoperably linked sequence. “Operably linked” means joined as part of thesame nucleic acid molecule, suitably positioned and oriented fortranscription to be initiated from the promoter.

Stringent Hybridization Conditions

“Stringent hybridization conditions” as used herein mean conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionsmay be selected to be about 5-10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) may be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probe molecules complementaryto the target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions may be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times stronger than background hybridization. Exemplarystringent hybridization conditions include the following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

Substantially Complementary

“Substantially complementary” as used herein means that a first sequenceis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the complement of a second sequence over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides,or that the two sequences hybridize under stringent hybridizationconditions.

Substantially Identical

“Substantially identical” as used herein means that a first and a secondsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respectto nucleic acids, if the first sequence is substantially complementaryto the complement of the second sequence.

Target Nucleic Acid

The target nucleic acid may comprise a target miRNA binding site or avariant thereof. One or more probes may bind the target nucleic acid.The target binding site has a perfect or near-perfect complementarity toone of the miRNA sequences as listed in Sanger database, version 10.1 orthe database of Molnar et al, 2007.

Variant

“Variant” as used herein referring to a nucleic acid means (i) a portionof a referenced nucleotide sequence; (ii) the complement of a referencednucleotide sequence or portion thereof; (iii) a nucleic acid that issubstantially identical to a referenced nucleic acid or the complementthereof; or (iv) a nucleic acid that hybridizes under stringentconditions to the referenced nucleic acid, complement thereof, or asequence substantially identical thereto.

Wild Type Sequence

As used herein, the term “wild type sequence” refers to a coding, anon-coding or an interface sequence which is an allelic form of sequencethat performs the natural or normal function for that sequence. Wildtype sequences include multiple allelic forms of a cognate sequence, forexample, multiple alleles of a wild type sequence may encode silent orconservative changes to the protein sequence that a coding sequenceencodes.

2. Nucleic Acids

“Nucleic acid” or “oligonucleotide” or “polynucleotide”, as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid may be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that may hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequences. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs may be included that may have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, which are incorporated herein by reference. Nucleic acidscontaining one or more non-naturally occurring or modified nucleotidesare also included within one definition of nucleic acids. The modifiednucleotide analog may be located for example at the 5′-end and/or the3′-end of the nucleic acid molecule. Representative examples ofnucleotide analogs may be selected from sugar- or backbone-modifiedribonucleotides. It should be noted, however, that alsonucleobase-modified ribonucleotides, i.e. ribonucleotides, containing anon-naturally occurring nucleobase instead of a naturally occurringnucleobase such as uridines or cytidines modified at the 5-position,e.g. 5-(2-amino) propyl uridine, 5-bromo uridine; adenosines andguanosines modified at the 8-position, e.g. 8-bromo guanosine; deazanucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides,e.g. N₆-methyl adenosine are suitable. The 2′-OH-group may be replacedby a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or CN,wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.Modified nucleotides also include nucleotides conjugated withcholesterol through, e.g., a hydroxyprolinol linkage as described inKrutzfeldt et al., Nature 438:685-689 (2005), Soutschek et al., Nature432:173-178 (2004), and U.S. Patent Publication No. 20050107325, whichare incorporated herein by reference. Additional modified nucleotidesand nucleic acids are described in U.S. Patent Publication No.20050182005, which is incorporated herein by reference. Modifications ofthe ribose-phosphate backbone may be done for a variety of reasons,e.g., to increase the stability and half-life of such molecules inphysiological environments, to enhance diffusion across cell membranes,or as probes on a biochip. The backbone modification may also enhanceresistance to degradation, such as in the harsh endocytic environment ofcells. Mixtures of naturally occurring nucleic acids and analogs may bemade; alternatively, mixtures of different nucleic acid analogs, andmixtures of naturally occurring nucleic acids and analogs may be made.

The nucleic acids provided herein comprise the sequences of SEQ ID NOS:1-88 and variants thereof. The variant may be a complement of thereferenced nucleotide sequence. The variant may also be a nucleotidesequence that is substantially identical to the referenced nucleotidesequence or the complement thereof. The variant may also be a nucleotidesequence which hybridizes under stringent conditions to the referencednucleotide sequence, complements thereof, or nucleotide sequencessubstantially identical thereto. Accordingly, SEQ ID NOS: 1-88, as wellas fragment thereof or sequences at least about 80% identical thereto,or sequences capable of hybridizing to, may be used to modify the oilcontent in a plant. Cells and transgenic plants produced by transductionof a vector comprising nucleic acids of the invention, are an additionalfeature of the invention.

The nucleic acid may have a length of from about 10 to about 250nucleotides. The nucleic acid may have a length of at least 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250nucleotides. The nucleic acid may be synthesized or expressed in a cell(in vitro or in vivo) using a synthetic gene. The nucleic acid may besynthesized as a single strand molecule and hybridized to asubstantially complementary nucleic acid to form a duplex. The nucleicacid may be introduced to a cell, tissue or organ in a single- ordouble-stranded form or capable of being expressed by a synthetic geneusing methods well known to those skilled in the art, including asdescribed in U.S. Pat. No. 6,506,559 which is incorporated by reference.

Also contemplated are uses of nucleic acid sequences, also referred toherein as oligonucleotides or polynucleotides, typically having at least12 bases, preferably at least 15, more preferably at least 20, 30, or 50bases, which hybridize under at least highly stringent (or ultra-highstringent or ultra-ultra-high stringent conditions) conditions to apolynucleotide sequence described above. Subsequences of the nucleicacid sequences of the invention, including polynucleotide fragments andoligonucleotides are useful as nucleic acid probes and primers. Anoligonucleotide suitable for use as a probe or primer is at least about15 nucleotides in length, more often at least about 18 nucleotides,often at least about 21 nucleotides, frequently at least about 30nucleotides, or about 40 nucleotides, or more in length. A nucleic acidprobe is useful in hybridization protocols, e.g., to identify additionalnucleic acid sequences homologs of the invention, including protocolsfor microarray experiments. Primers can be annealed to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, and then extended along the targetDNA strand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods.

2a. MicroRNAs their Processing

MicroRNAs (or miRNAs) are 17 to 24 nucleotide silencing RNAs, processedfrom a hairpin stem-loop region of a longer transcript precursor, byDicer-like enzymes.

Plant miRNAs may be found in genomic regions not associated with proteincoding genes. Transcription of a gene encoding miRNA genarates apri-miRNA which is processed by DCL1, optionally with the aid of HYL1and other factors, to a miRNA:miRNA* duplex which may include 5′phosphates (P) and two-nucleotide 3′ overhangs. DCL1 cuts preferentiallyat specific positions in the miRNA stem-loop precursor. (Pre-miRNAs,which are readily detectable in animals, appear to be short-lived inplants).

The 3′ sugars of the miRNA:miRNA* duplex may be methylated possiblywithin the nucleus. The miRNA is exported to the cytoplasm by HST,probably with the aid of additional factors. The miRNA strand ispreferentially loaded into the RNA induced silencing complex (RISC),where it is protected from degradation, whereas the miRNA* strand ispreferentially excluded from the silencing complex and consequentiallysubject to degradation. Accordingly, the final product of the miRNAbiogenesis pathway is a single-stranded RNA incorporated into asilencing complex.

Plant miRNAs may facilitate both target cleavage and translationalrepression. Plant miRNAs may guide the cleavage of complementary ornearly complementary mRNAs, optionally by negatively regulate stabilityof their targets. Cleavage may occur between the target nucleotides thatpair to nucleotides 10 and 11 of the miRNA.

Plant miRNAs may occur in gene families, each family contains severalloci within a single genome, each potentially encoding identical ornearly identical mature miRNAs.

The nucleic acids of this invention may comprise a sequence of a miRNA(including miRNA*) or a variant thereof. The miRNA sequence may comprisefrom 13-33, 18-24 or 21-23 nucleotides. The miRNA may also comprise atotal of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39 or 40 nucleotides. The sequence of the miRNA may be the first13-33 nucleotides of the precursor. The sequence of the miRNA may alsobe the last 13-33 nucleotides of the precursor. The sequence of themiRNA may comprise the sequence of SEQ ID NOS: 1-39, 71-73 and 84 orvariants thereof. The sequence of the microRNA precursor may comprisethe sequence of SEQ ID NOS: 40-70, 74-75 and 85 or variants thereof, asdetailed in Table 1.

TABLE 1 Details regarding miRs associated with oil content in C.reinhardtii and their corresponding hairpins miR Locus Position inHairpin SEQ ID NO Name¹ Accession ID genome SEQ ID NO 1 cre-miR905MI00605698 N/A scaffold 75: 40 370850-371142 2 cre-miR908.1 MI0005701N/A scaffold 213: 41 20092-20321 3 cre-miR908.2 MI0005701 N/A scaffold213: 41 20092-20322 4 cre-miR908.3 MI0005701 N/A scaffold 213: 4120092-20323 5 cre-miR910 MI0005703 N/A scaffold 17: 42 966108-966334 6cre-miR916 MI0005711 N/A scaffold 86: 43 94576-95507 7 cre-miR919.1MI0005709 N/A scaffold 6: 44 1014120-1014384 8 cre-miR919.2 MI0005709N/A scaffold 6: 44 1014120-1014384 9 cre-miR1145.1 MI0006206 N/Ascaffold 2: 45 546495-546515 10 cre-miR1145.2 MI0006206 N/A scaffold2:45 546542-546562 11 cre-miR1149.1 MI0006210 N/A scaffold 1: 465256729-5256842 12 cre-miR1149.2 MI0006210 N/A scaffold 1: 465256729-5256842 13 cre-miR1151a MI0006212 N/A scaffold 10: 471934033-1934142 14 cre-miR1151b MI0006213 N/A scaffold 10: 481935781-1935924 15 cre-miR1152 MI0006214 N/A scaffold 12: 491998777-1998906 16 cre-miR1155 MI0006217 N/A scaffold 15: 502058507-2058606 17 cre-miR1157 MI0006219 N/A scaffold 18: 51251268-251404 18 cre-miR1160.1 MI0006221 N/A scaffold 26: 52727941-728377 19 cre-miR1160.2 MI0006221 N/A scaffold 26: 52727941-728377 20 cre-miR1160.3 MI0006221 N/A scaffold 26: 52727941-728377 21 cre-miR1167 MI0006234 N/A scaffold 10: 532068926-2069014 22 cre-miR1168.1 MI0006228 N/A scaffold 40: 54432487-432679 23 cre-miR1168.2 MI0006228 N/A scaffold 40: 54432487-432679 24 cre-miR1169 MI0006229 N/A scaffold 46: 55 613349-61344425 cre-miR1171 MI0006231 N/A scaffold 51: 56 419396-419582 26 CRMIR5 N/Acre.02466 scaffold 35: 57 929861-929986 27 CRMIR15 N/A cre.02674scaffold 558: 58 7360-8065 28 CRMIR16 N/A cre.02674 scaffold 558: 597360-8065 29 CRMIR29 N/A cre.02714 scaffold 6: 60 118475-118760 30CRMIR47 N/A cre.01795 scaffold 1: 61 186381-186552 31 CRMIR72 N/Acre.01824 scaffold 1: 62 6352149-6352516 32 CRMIR96 N/A cre.01854scaffold 10: 63 2282657-2282780 33 CRMIR100 N/A cre.02194 scaffold 2: 643204761-3204858 34 CRMIR101 N/A cre.02194 scaffold 2: 65 3204761-320485835 CRMIR121 N/A cre.02790 scaffold 68: 66 317493-317595 36 CRMIR123 N/Acre.02790 scaffold 68: 67 317605..317763 37 CRMIR129 N/A cre.02862scaffold 75: 68 284481-284798 38 CRMIR152 N/A cre.03680 TIN168528_x1: 6964..367 39 CRMIR156 N/A cre.04503 TIN455283_x1: 70 587..648 71cre-miR1142 MI0006203 NA scaffold 34: 74 1054527-1055286 72 CRMIR136 N/A02884 scaffold 80: 75 14307-15220 73 CRMIR137 N/A 02884 scaffold 80: 7514307-15220 84 CRMIR167 N/A cre.05507 scaffold 17: 85 1800973-1801124¹The miRs of SEQ ID NOS: 1-25, 71 and their corresponding hairpins (SEQID NOS: 40-56, 74) appear in the Sanger database, version 10.1.Sequences 26-39, 72-73, 84 and their corresponding hairpins (SEQ ID NOS:57-70, 75, 85) are in accordance with the database of Molnar et al,2007.3. Transgenic Plants

A transgenic plant as used herein refers to a plant that containsgenetic material not found in a wild-type plant of the same species,variety or cultivar. The genetic material may include a transgene, aninsertional mutagenesis event (such as by transposon or T-DNAinsertional mutagenesis), an activation tagging sequence, a mutatedsequence, a homologous recombination event or a sequence modified bychimeraplasty. Typically, the foreign genetic material has beenintroduced into the plant by human manipulation, but any method can beused as one of skill in the art recognizes. A transgenic plant maycontain an expression vector or cassette.

Successful examples of the modification of plant characteristics bytransformation with cloned sequences which serve to illustrate thecurrent knowledge in this field of technology, and which are hereinincorporated by reference, include U.S. Pat. Nos. 6,235,529, 7,429,691and 7,256,283.

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practicing the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantgrowth.

Following transformation, plants are preferably selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or herbicide resistanceon the transformed plants and selection of transformants can beaccomplished by exposing the plants to appropriate concentrations of theantibiotic or herbicide.

After transformed plants are selected and grown to maturity, thoseplants showing a modified trait are identified. The modified trait canbe enhanced oil content.

Additionally, to confirm that the modified trait is due to changes inexpression levels or activity of nucleic acid sequences of theinvention, mRNA expression can be analyzed by using Northern blots,RT-PCR or microarrays.

3.1 Transgenic miR-based Enhancement of Oil Yield in Plants' Seeds

In order to enhance the oil content in plants' seeds according to someaspects of the invention, various varieties of plants differing in oilcontent, of Canola, soybean, Jatropha, camelina, corn and castor bean(widely used for biodiesel production) are obtained, and throughouttheir various developmental stages, including floral tissue and youngseeds, RNA is extracted and microRNA expression profiles are created andcompared.

For example, two corn varieties, named sweet and super-sweet (bothobtained from Galil Seeds—Israel), have oil contents of 7% and 18%respectively. RNA is extracted from four days old seedlings of these twovarieties, and microarray analysis is performed by LC Sciences (Austin,Tex.). The microRNA expression profiles of these varieties are compared.

Differentially expressed microRNAs related to seed oil content areidentified and validated by qPCR. Plants are transgenically transformedto overexpress microRNA precursors or to express microRNA-resistanttargets. Transgenic plants are grown parallel to corresponding wild-typeplants, and genetically transformed plants with relatively enhanced seedoil content are identified.

3.2 Algal Technology

Although there are thousands of species of known naturally occurringalgae, any one may be used for biodiesel production according to someembodiments of the invention. The skilled artisan will realize thatdifferent algal strains will exhibit different growth and oilproductivity and that under different conditions the system may containa single strain of algae or a mixture of strains with differentproperties, or strains of algae plus symbotic bacteria. The algalspecies used may be optimized for geographic location, temperaturesensitivity, light intensity, pH sensitivity, salinity, water quality,nutrient availability, seasonal differences in temperature or light, thedesired end products to be obtained from the algae and a variety ofother factors.

3.3 Genetic Modification of Algae

The genetic modification of algae for specific product outputs isrelatively straight forward using techniques well known in the art. Incertain embodiments, algae of use to produce biodiesel may begenetically engineered (transgenic) to contain one or more isolatednucleic acid sequences that enhance oil production or provide othercharacteristics of use for algal culture, growth, harvesting or use.Methods of stably transforming algal species and compositions comprisingisolated nucleic acids of use are well known in the art and any suchmethods and compositions may be used in the practice of the presentinvention. Exemplary transformation methods of use may includemicroprojectile bombardment, electroporation, protoplast fusion,PEG-mediated transformation, DNA-coated silicon carbide whiskers or useof viral mediated transformation (see, e.g., Sanford et al., 1993, Meth.Enzymol. 217:483-509; Dunahay et al., 1997, Meth. Molec. Biol. 62:503-9,incorporated herein by reference).

U.S. Pat. No. 5,661,017 discloses methods for algal transformation ofchlorophyll C-containing algae, such as the Bacillariophyceae,Chrysophyceae, Phaeophyceae, Xanthophyceae, Raphidophyceae,Prymnesiophyceae, Cryptophyceae, Cyclotella, Navicula, Cylindrotheca,Phaeodactylum, Amphora, Chaetoceros, Nitzschia or Thalassiosira.

Following transformation, algae are preferably selected using a dominantselectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic or other resistance onthe transformed algae and selection of transformants can be accomplishedby exposing the algae to appropriate concentrations of the selectablemarker to select for transformed algae. Selectable markers of use mayinclude, neomycin phosphotransferase, aminoglycoside phosphotransferase,aminoglycoside acetyltransferase, aminoglycoside-O-phosphotransferase,chloramphenicol acetyl transferase, hygromycin B phosphotransferase,bleomycin binding protein, phosphinothricin acetyltransferase,bromoxynil nitrilase, glyphosate-resistant5-enolpyruvylshikimate-3-phosphate synthase, cryptopleurine-resistantribosomal protein S 14, emetine-resistant ribosomal protein S 14,sulfonylurea-resistant acetolactate synthase, imidazolinone-resistantacetolactate synthase, streptomycin-resistant 16S ribosomal RNA,spectinomycin-resistant 16S ribosomal RNA, erythromycin-resistant 23 Sribosomal RNA or methyl benzimidazole-resistant tubulin. Regulatorynucleic acid sequences to enhance expression of a transgene are known,such as C. cryptica acetyl-CoA carboxylase 5′-untranslated regulatorycontrol sequence, a C. cryptica acetyl-CoA carboxylase 3′-untranslatedregulatory control sequence, and combinations thereof.

Additional possible selection is via auxotroph mutants. The selectablemarker is then a biosynthetic gene allowing the mutant to grow in theabsence of the substance they are auxotroph to. This includes theability to synthesize amino acids, vitamins and other essentialco-factors. A specific example is the ARG7 locus that can rescue thearg2 mutation in C. reinhardtii (Debuchy et al. (1989) EMBO J. 8,2803-2809) and allow cells to grow in the absence of the amino acidarginine.

3.4 Separation of Algae and Extraction of Oil

In various embodiments, algae may be separated from the medium, andvarious algal components, such as oil, may be extracted using any methodknown in the art. For example, algae may be partially separated from themedium using a standing whirlpool circulation, harvesting vortex and/orsipper tubes, as discussed below. Alternatively, industrial scalecommercial centrifuges of large volume capacity may be used tosupplement or in place of other separation methods. Such centrifuges maybe obtained from known commercial sources (e.g., Cimbria Sket or IBGMonforts, Germany; Alfa Laval AJS, Denmark). Centrifugation,sedimentation and/or filtering may also be of use to purify oil fromother algal components. Separation of algae from the aqueous medium maybe facilitated by addition of flocculants, such as clay (e.g., particlesize less than 2 microns), aluminum sulfate or polyacrylamide. In thepresence of flocculants, algae may be separated by simple gravitationalsettling, or may be more easily separated by centrifugation.

The skilled artisan will realize that any method known in the art forseparating cells, such as algae, from liquid medium may be utilized. Forexample, U.S. Pat. No. 6,524,486, incorporated herein by reference,discloses a tangential flow filter device and apparatus for partiallyseparating algae from an aqueous medium. Other published methods foralgal separation and/or extraction may also be used. (See, e.g., Rose etal., Water Science and Technology 1992, 25:319-327; Smith et al.,Northwest Science, 1968, 42:165-171; Moulton et al., Hydrobiologia 1990,204/205:401-408; Borowitzka et al., Bulletin of Marine Science, 1990,47:244-252; Honeycutt, Biotechnology and Bioengineering Symp. 1983,13:567-575).

In various embodiments, algae maybe disrupted to facilitate separationof oil and other components. Any method known for cell disruption may beutilized, such as ultrasonication, French press, osmotic shock,mechanical shear force, cold press, thermal shock, rotor-statordisruptors, valve-type processors, fixed geometry processors, nitrogendecompression or any other known method. High capacity commercial celldisruptors may be purchased from known sources. (E.g., GEA Niro Inc.,Columbia, Md.; Constant Systems Ltd., Daventry, England; Microfluidics,Newton, Mass.).

4. Promoters

Promoters are expressed in many, if not all, cell types of many plants.Promoters including those that are developmentally regulated orinducible may be used. For example, if it is necessary to silence thetarget gene specifically in a particular cell type the construct may beassembled with a promoter that drives transcription only in that celltype. Similarly, if the target gene is to be silenced following adefined external stimulus the construct may incorporate a promoter thatis be activated specifically by that stimulus. Promoters that are bothtissue specific and inducible by specific stimuli may be used.

A variety of plant gene promoters that regulate gene expression inresponse to environmental, hormonal, chemical, developmental signals,and in a tissue-active manner can be used in plants. Choice of apromoter is based largely on the phenotype of interest and is determinedby such factors as tissue (e.g., seed, fruit, root, pollen, vasculartissue, flower, carpel, etc.), inducibility (e.g., in response towounding, heat, cold, drought, light, pathogens, etc.), timing,developmental stage, and the like. Numerous known promoters have beencharacterized and can favorably be employed to promote expression of apolynucleotide of the invention in a transgenic plant or cell ofinterest.

5. Biochip

A biochip is also provided. The biochip may comprise a solid substratecomprising an attached probe or plurality of probes described herein.The probes may be capable of hybridizing to a target sequence understringent hybridization conditions. The probes may be attached atspatially defined addresses on the substrate. More than one probe pertarget sequence may be used, with either overlapping probes or probes todifferent sections of a particular target sequence. The probes mayeither be synthesized first, with subsequent attachment to the biochip,or may be directly synthesized on the biochip.

The solid substrate may be a material that may be modified to containdiscrete individual sites appropriate for the attachment or associationof the probes and is amenable to at least one detection method.Representative examples of substrates include glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses andplastics. The substrates may allow optical detection without appreciablyfluorescing.

The substrate may be planar, although other configurations of substratesmay be used as well. For example, probes may be placed on the insidesurface of a tube, for flow-through sample analysis to minimize samplevolume. Similarly, the substrate may be flexible, such as flexible foam,including closed cell foams made of particular plastics.

The biochip and the probe may be derivatized with chemical functionalgroups for subsequent attachment of the two. For example, the biochipmay be derivatized with a chemical functional group including, but notlimited to, amino groups, carboxyl groups, oxo groups or thiol groups.Using these functional groups, the probes may be attached usingfunctional groups on the probes either directly or indirectly using alinker. The probes may be attached to the solid support by either the 5′terminus, 3′ terminus, or via an internal nucleotide.

The probe may also be attached to the solid support non-covalently. Forexample, biotinylated oligonucleotides can be made, which may bind tosurfaces covalently coated with streptavidin, resulting in attachment.Alternatively, probes may be synthesized on the surface using techniquessuch as photopolymerization and photolithography.

6. Expression Vector

The expression vector or cassette typically comprises an encodingsequence operably linked (i.e., under regulatory control of) toappropriate inducible or constitutive regulatory sequences that allowfor the expression of polypeptide. The expression vector can beintroduced into a plant by transformation or by breeding aftertransformation of a parent plant. A plant refers to a whole plant aswell as to a plant part, such as seed, fruit, leaf, or root, planttissue, plant cells or any other plant material, e.g., a plant explant,as well as to progeny thereof, and to in vitro systems that mimicbiochemical or cellular components or processes in a cell.

A number of expression vectors suitable for stable transformation ofplant cells or for the establishment of transgenic plants have beendescribed including those described in Weissbach and Weissbach (1989)Methods for Plant Molecular Biology, Academic Press, and Gelvin et al.(1990) Plant Molecular Biology Manual, Kluwer Academic Publishers.Specific examples include those derived from a Ti plasmid ofAgrobacterium tumefaciens, as well as those disclosed byHerrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) NucleicAcids Res. 12: 8711-8721, Klee (1985) Bio/Technology 3: 637-642, fordicotyledonous plants.

Alternatively, non-Ti vectors can be used to transfer the DNA intomonocotyledonous plants and cells by using free DNA delivery techniques.Such methods can involve, for example, the use of liposomes,electroporation, microprojectile bombardment, silicon carbide whiskers,and viruses. By using these methods transgenic plants such as wheat,rice (Christou (1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm(1990) Plant Cell 2: 603-618) can be produced. An immature embryo canalso be a good target tissue for monocots for direct DNA deliverytechniques by using the particle gun (Weeks et al. (1993) Plant Physiol.102: 1077-1084; Vasil (1993) Bio/Technology 10: 667-674; Wan and Lemeaux(1994) Plant Physiol. 104: 37-48, and for Agrobacterium-mediated DNAtransfer (Ishida et al. (1996) Nature Biotechnol. 14: 745-750).

Typically, plant transformation vectors include one or more cloned plantcoding sequence (genomic or cDNA) under the transcriptional control of5′ and 3′ regulatory sequences and a dominant selectable marker. Suchplant transformation vectors typically also contain a promoter, atranscription initiation start site, an RNA processing signal (such asintron splice sites), a transcription termination site, and/or apolyadenylation signal.

EXAMPLES Example 1 Materials and Methods

-   a. Algal material: The Chlamydomonas reinhardtii UTEX 90 strain    (University of Texas algae collection, Austin Tex.) was used in all    experiments except for transformation experiments where the cell    wall-less mutant cw15 (Chlamydomonas Center, Duke University) was    used. Algae were maintained in TAP medium in an Adaptis growth    chamber (Conviron, Canada) at 22° C. under constant white light at    an intensity of 175 mmol photon M⁻² s⁻¹ while being shaken at 120    rpm in 250-ml flasks containing 50 ml of growth medium.    -   Nitrogen starvation experiments were performed using either        Bristol medium (NaNO₃-2.94 mM, CaCl₂.2H₂O-0.17 mM,        MgSO₄.7H₂O-0.3 mM, K₂HPO₄.0.43 mM, KH₂PO₄-1.29 mM, NaCl-0.43 mM)        or Bristol medium depleted of NaNO₃, for nitrate-limiting        conditions.-   b. Database: MicroRNA sequences were derived from the 10.1 version    of miRBase released December 2007 and consists of the 673    non-redundant sequences defined as “Viridiplantae” and the 47    sequences submitted for C. reinhardtii. Additionally, 170    recently-identified putative microRNA sequences by present in the C.    reinhardtii silencing RNA database were included.-   c. Microarray design: Custom microarrays were manufactured by    Agilent Technologies by in situ synthesizing DNA oligonucleotide    probes for 890 plant and algal microRNAs with each probe being    printed in triplicate.    -   Fourteen negative control probes were designed using the sense        sequences of different microRNAs, chosen from different plants        and algae. Two groups of positive control probes were used: (i)        synthetic small RNA that were spiked to the RNA before labeling        to verify the labeling efficiency, and (ii) probes for abundant        small nuclear RNAs (U1, U2, U3, U4, U5, U6, for three plant        species and for C. reinhardtii) were spotted on the array to        verify RNA's quantity and quality.-   d. Sample labeling: Five μg of total RNA were labeled by ligation of    an RNA-linker, p-rCrU-Cy/dye (Eurogentec), to the 3′-end with Cy3 or    Cy5. The labeling reaction contained total RNA, spikes (0.1-20    fmoles), 300-400 ng RNA-linker-dye, 15% DMSO, 1× ligase buffer and    20 units of T4 RNA ligase (NEB) and proceeded at 4° C. for 1 hr    followed by 1 hr at 37° C. on PCR machine.-   e. Microarray hybridization and scanning: Five micrograms of total    RNA were labeled by ligation of Cy3 or Cy5 to the 3′-end. The    labeled RNA was mixed with 3× hybridization buffer (Ambion, Austin    Tex.) and hybridized with the slides for 12-16 hrs in an Agilent    Rotational oven at 10-13 rpm. Following the hybridization, the    arrays were washed twice in room temperature with 1×SSC and 0.2%    SDS. Next, the arrays were washed in 0.1×SSC followed by a 1 min    wash in fresh Agilent Stabilization and Drying solution (Agilent,    Santa Clara Calif.).-   f. Array signal calculation and normalization: Array images were    analyzed using the Feature Extraction software (FE) 9.5.1 (Agilent,    Santa Clara Calif.). Experiments were repeated four times and    triplicate spots were combined to produce one signal for each probe    by taking the logarithmic mean of reliable spots. All data was    log-transformed (natural base) and the analysis was performed in    log-space. A reference data vector for normalization R was    calculated by taking the median expression level for each probe    across nitrogen starvation samples. For each sample data vector S, a    2nd degree polynomial F was found so as to provide the best fit    between the sample data and the reference data, such that R≈F(S).    For each probe in the sample (element Si in the vector S), the    normalized value (in log-space) Mi was calculated from the initial    value Si by transforming it with the polynomial function F, so that    Mi=F(Si). P-values were calculated using a two-sided t-test on the    log-transformed normalized fluorescence signal. For each microRNA    the fold-difference (ratio of the median normalized fluorescence)    was calculated.

Example 2 Oil Biosynthesis Induced by Nitrogen Starvation and StrongLight, and Differentially Expressed microRNAs Related Thereto

2.1 Induction of Oil Biosynthesis in C. reinhardtii

The oil content in C. reinhardtii was monitored under two different setsof conditions: nitrogen starvation and strong light.

-   Nitrogen starvation: C. reinhardtii cells were grown to logarithmic    phase in algae culture broth and 50-ml aliquots were collected by    centrifugation at 3,220×g for 1 minute. Cells were washed once in 25    ml of either Bristol (for nitrogen sufficient conditions) or Bristol    medium without NaNO₃ (Bristol-N₂) (for nitrogen limiting conditions)    and resuspended in 50 ml of either Bristol or Bristol-N₂ medium    respectively. Cells were shaken at 120 rpm at 22° C. under constant    light and were harvested after 24 hrs.-   Light induction: C. reinhardtii cells were grown to logarithmic    phase under standard light conditions (1×18 Watts white fluorescent    lamp). For oil induction the standard light was supplemented with    4×36 Watts lamps.

Cells were harvested after 6, 16, 24 and 48 hours, and the oil contentof control and treated cells was performed using Nile Red staining andmeasuring the fluorescence. 200 μl of algal cells were placed in a96-well plate (Costar 3596), mixed with 10 μl of Nile Red solution(N3013, Sigma. 1 mg/ml stock in 2-propanol) and incubated for 15 minutesat room-temperature. Fluorescence was measured using a Thermo ElectronFluoroskan Ascent with excitation at 544 nm and emission at 590 nm 200μl of fresh growth media with 20 μl of Nile Red were used for blankmeasurements. Fluorescence was normalized against cell density asmeasured by an ELx808 microplate reader (BIOTEK Instruments Inc.) at 450nm

As indicated in FIG. 1, the kinetics of oil content induction was foundto be different for nitrogen starvation and strong light. Nitrogenstarvation caused a noticeable induction already after 16 hours with apeak at 24 hours, whereas induction by strong light was only noticedafter 24 hours but seems to grow stronger over time at least until 48hours.

2.2 Differentially Expressed microRNAs

Nitrogen starvation experiments were performed by growing C. reinhardtiito logarithmic phase, washing off traces of nitrogen from the growthmedium and divided into two treatments containing Bristol medium with orwithout NaNO₃ as a nitrogen source. Cells were grown for 16, 24 and 48hours after which RNA was extracted and used for microRNA arrayanalysis. Similarly, RNA was extracted from C. reinhardtii cells thatwere grown to logarithmic phase under standard light or strong light.The RNA was extracted after 6, 16 or 24 hours and was used for microRNAarray analysis.

Total RNA was extracted using the mirVana™ kit (Ambion, Austin Tex.)following the instruction manual for total RNA isolation protocol usingthe following adaptations: 50 ml of alga cells were harvested bycentrifugation at 4,000 rpm for 1 minute, washed once with 25 ml of coldalgae culture broth medium and re-suspended in 2 ml of the mirVana™ kitLysis/Binding Buffer. Cells were ground using a Polytron PT-MR 2100homogenizer (Kinematica AG, Switzerland).

Expression levels of microRNAs in each of the treatments were measuredas described in Materials and Methods section.

MicroRNAs upregulated and downregulated in C. reinhardtii under nitrogenstarvation are shown in Tables 2a and 2b respectively. MicroRNAsupregulated and downregulated in response to strong light are shown inTables 3a and 3b respectively.

TABLE 2: MicroRNAs Up-regulated (a) and Down-regulated (b) in Responseto Nitrogen Starvation (Bristol-N₂).

Only microRNAs demonstrating a minimum fold change of 1.5 in (a) or 0.6in (b) in at least one of the time points are shown. Normalizedexpression values are shown in log scale.

TABLE 2a MicroRNAs upregulated in response to nitrogen starvation. SEQ16 hours: 24 hours: 48 hours: ID Fold Fold Fold MicroRNA NO −N₂ +N₂Change −N₂ +N₂ Change −N₂ +N₂ Change CRMIR29 29 8.1 7.7 1.4 8.5 7.8 1.68.1 8.1 1.0 CRMIR47 30 12.0 12.1 1.0 12.3 12.3 1.0 12.7 12.1 1.5 cre- 48.4 8.5 1.0 8.3 8.3 1.0 8.8 8.0 1.8 miR908.3 cre-miR916 6 12.8 12.4 1.410.1 9.4 1.7 12.2 11.9 1.3 cre- 12 7.3 7.1 1.1 7.8 7.1 1.5 7.8 7.7 1.0miR1149.2 cre- 20 8.5 8.6 0.9 8.1 7.8 1.3 8.6 8.0 1.5 miR1160.3 cre- 2510.0 9.9 1.1 9.4 9.5 1.0 10.1 9.5 1.5 miR1171

TABLE 2b MicroRNAs downregulated in response to nitrogen starvation. SEQ16 hours: 24 hours: 48 hours: ID Fold Fold Fold MicroRNA NO −N₂ +N₂Change −N₂ +N₂ Change −N₂ +N₂ Change CRMIR15 27 8.2 8.2 1.0 5.6 5.6 1.06.4 7.5 0.5 CRMIR16 28 7.6 7.7 1.0 5.6 5.6 1.0 5.9 7.1 0.4 CRMIR96 3210.8 10.8 1.0 10.7 10.9 0.9 10.4 11.0 0.7 CRMIR129 37 10.4 10.4 1.0 9.510.2 0.6 10.1 10.2 0.9 CRMIR152 38 8.4 8.5 0.9 5.6 5.6 1.0 6.8 7.8 0.5cre-miR910 5 9.6 9.9 0.9 9.9 10.4 0.7 10.7 11.7 0.5 cre- 16 13.3 13.11.1 12.2 12.9 0.6 12.9 13.0 0.9 miR1155 cre- 17 11.5 11.5 0.9 11.9 12.50.7 11.9 11.7 1.1 miR1157 cre- 23 11.6 11.8 0.9 10.3 10.9 0.6 11.2 11.31.0 miR1168.2Table 3: MicroRNAs Upregulated (a) and Down-regulated (b) in Response toStrong Light. Normalized expression values are shown in log scale.

TABLE 3a MicroRNAs up-regulated in response to strong light. SEQ Stronglight ID Standard Fold 16 Fold 24 Fold MicroRNA NO: light 6 hours changehours change hours change CRMIR5 26 12.30 13.05 1.62 13.40 2.00 12.301.07 CRMIR72 31 11.59 12.10 1.42 12.17 1.49 12.12 1.44 CRMIR101 34 9.189.88 1.62 10.38 2.29 10.05 1.82 CRMIR121 35 6.94 7.54 1.52 7.66 1.657.36 1.34 cre-miR919.2 8 11.80 12.59 1.72 12.34 1.45 12.96 2.23cre-miR1151a 13 12.83 13.69 1.82 13.81 1.97 13.84 2.01 cre-miR1151b 1412.84 13.78 1.92 13.73 1.85 13.83 1.99 cre-miR1152 15 11.61 12.86 2.3912.25 1.56 12.12 1.43 cre-1167 21 9.73 ND ND 11.04 1.3 ND ND cre-miR116924 10.51 11.20 1.61 11.43 1.90 11.53 2.03 CRMIR167 84 10.2 10.4 1.2611.1 1.8 11.0 1.6 ND: Not Detected

TABLE 3b MicroRNAs and downregulated in response to strong light. SEQStrong light ID Standard Fold 16 Fold 24 Fold MicroRNA NO: light 6 hourschange hours change hours change CRMIR15 27 9.69 6.95 0.15 6.53 0.116.20 0.09 CRMIR16 28 9.00 6.57 0.19 6.33 0.16 6.06 0.13 CRMIR152 3810.19 7.12 0.12 6.98 0.11 6.62 0.08 CRMIR156 39 7.82 6.87 0.52 7.00 0.576.83 0.50 cre-miR905 1 9.40 8.54 0.55 8.59 0.57 8.13 0.41 cre-miR1145.19 7.93 7.25 0.62 7.10 0.56 6.85 0.47

Example 3 Oil Biosynthesis Related to Nitrate-limiting Conditions, andDifferentially Expressed microRNAs Related Thereto

3.1 Induction of Oil Biosynthesis in C. reinhardtii

C. reinhardtii cells were grown to logarithmic phase in TAP medium and50 ml aliquots were collected by centrifugation at 3,220×g for 1 minute.Cells were washed once in 25 ml of either Bristol medium(nitrate-sufficient conditions) or nitrate-free Bristol medium(nitrate-limiting conditions) and resuspended in 50 ml of either Bristolor nitrate-free Bristol medium respectively. Cells were shaken at 120rpm at 22° C. and were harvested after 24 hrs.

Relative evaluation of neutral lipids content was performed using NileRed staining, with some minor modifications: 200 μl of algal cells wereplaced in a 96-well plate (Costar 3596), mixed with 10 μl of Nile Redsolution (N3013, Sigma. 1 mg/ml stock in acetone) and incubated for 10minutes at room-temperature. Fluorescence was measured using a ThermoElectron Fluoroskan Ascent with excitation at 485 nm and emission at 590nm, which were previously found to detect mainly neutral lipids. 200 μlof fresh growth media with 10 μl of Nile Red were used for blankmeasurements. Fluorescence was normalized against cell density asmeasured by an ELx808 microplate reader (BIOTEK Instruments Inc) at 630nm and verified by cell count of selected samples. The significance ofthe change in lipid content was calculated by using one sided student'st-test. The relative changes in lipid content between different sampleswere verified by the gravimetric method, performed by the MylnefieldLipid Analysis laboratory (Dundee, Scotland).

Neutral lipid content was found to increase in nitrate-limitingconditions by 33% as measured by nile-red staining (p value=0.02) and by24% by the gravimetric method.

3.2 Differentially Expressed microRNAs

Total RNA was extracted using the mirVana™ kit (Ambion, Austin Tex.)following the instruction manual. Samples were DNAse-treated (Ambion,Austin Tex.) for 2 hours, phenol-treated and ethanol-precipitated. TotalRNA concentration was determined using an ND-1000 NanoDropspectrophotometer (Thermo Scientific, Waltham Mass.).

Expression levels of microRNAs in C. reinhardtii cells grown undernitrate-sufficient or nitrate-limiting conditions were measured asdescribed in example 2. Comparing the expression levels, five microRNAswere identified that were differentially expressed in these conditions,with a p value≦0.05 and a minimum fold change of 1.5. As detailed intable 4 below, all of the differentially-expressed microRNAs were foundto be downregulated in nitrate-limiting relative to nitrate-sufficientconditions.

TABLE 4 Differentially expressed microRNAs in nitrate-sufficientrelative to nitrate- limiting conditions MicroRNA name SEQ ID NO: Foldchange p value cre-miR1142 71 1.6 0.04 cre-miR1167 21 1.6 0.03 CRMIR12937 1.5 0.02 CRMIR136 72 1.5 0.02 CRMIR137 73 1.9 0.02

The differential expression of microRNAs in nitrate-limiting relative tonitrate-sufficient conditions is further exemplified in the scatterplotof FIG. 3, and in the boxplots of FIGS. 4A-4E.

Expression of CRMIR136 (SEQ ID NO: 72) was validated by qRT-PCR asfollows: Briefly, RNA was incubated in the presence of a poly Apolymerase enzyme (Takara, Otsu Japan), MnCl₂, and ATP for 1 h at 37° C.Then, using an oligo dT primer harboring a consensus sequence, reversetranscription was performed on total RNA using SuperScript II RT(Invitrogen, Carlsbad Calif.). Next, the cDNA was amplified by real timePCR; this reaction contained a microRNA-specific forward primer and auniversal reverse primer complementary to the consensus 3′ sequence ofthe oligo dT tail. Results represent the median of four repeats witheach one done in triplicate and the signal was normalized against themedian of three reference microRNAs. The sequences used in the PCRprocedure are detailed in table 5 below:

TABLE 5 Sequences used in PCR validation of CRMIR136(SEQ ID NO: 72) expression SEQ Role in PCR Sequence ID NO:oligo dT primer GCGAGCACAGAATTAATACGACT 79 CACTATCGGTTTTTTTTTTTTVN¹microRNA-specific forward primer TGCGGGATCCAGTGCTGG 76universal reverse primer GCGAGCACAGAATTAATACGAC 77 ¹V represents A, G orC, and N represents any nucleotide.

Accordingly, the differential expression of CRMIR136 (SEQ ID NO: 72) wasvalidated by qRT-PCR, as it was found to be downregulated innitrate-limiting relative to nitrate-sufficient conditions with afold-change of 1.5 (p value=0.01), which correlates with the fold changeof 1.5 identified by the microarray experiments.

Example 4 Oil Content in Transformed C. reinhardtii Cells

Synthetic DNA clones were synthesized by Genscript. These clonescontained, respectively, the hairpins cre-mir1151b (SEQ ID NO: 48),cre-mir1157 (SEQ ID NO: 51), CRMIR121 (SEQ ID NO: 66). An additionalclone contained a sequence consisting of the mature microRNA sequence ofCRMIR5 inserted in the backbone of cre-mir1162 as follows:CATATGGCGGGGCCCTGACACCACTGCGGCCGTAGGACGGCTAGATCCGGGACCGCGCCTGGACCCGAGGGAGGACCCCTCGGGACCCGGTACGTCGTCCCGGATCTAGC,hereby designated SEQ ID NO: 78. (This artificial sequence, bearing themature microRNA sequence of CRMIR5, was created because the endogenoushairpin of CRMIR5 (SEQ ID NO: 57) comprises sequences of various maturemicroRNAs, and therefore is less suitable for transformation.)

The vectors were designed to contain an NdeI site at their 5′ end and anEcoRI site at their 3′ end and were constructed in the pUC19 vector. Thevectors were digested with NdeI and EcoRI to release the fragmentscontaining the indicated microRNAs, which was cloned to the pGenD-Blevector (N. Fischer & J. D. Rochaix, Molecular General Genomics, 2001,265:888-894). pGenD-Ble was previously digested with NdeI and EcoRI torelease the ble gene. The digested pGenD-Ble vector was separated by gelelectrophoresis and the fragment containing the vector minus the blegene was excised from the gel.

The resulting vector containing the pGenD promoter-microRNA hairpin-andpGenD terminator were designated pGenD-1151, pGenD-1157, pGenD-CR121 andpGenD-CR5 in1162. The vectors were co-transformed into C. reinhardtiicw15 together with pGenD-Aph following the method described by K Kindle(K. L. Kindle, Proc. Natl. Acad. Sci. U.S.A. 93 (1990) 13689-13693).PgenD-Aph was prepared by introducing the Aph gene (I. Sizova, M.Fuhrmann and P. Hegemann, Gene 277 (2001) 221-229) between the NdeI andEcoRI sites of pGenD-Ble as described above. Cells were selected on TAPmedium containing 15 microgram/nal paromomycin.

Cell cultures were grown in Bristol medium, and relative evaluation ofneutral lipids content was performed as described in example 3.1 above.

Table 6 below provides results of oil content evaluation in fourtransformed C. reinhardtii lines (several clones for each line)overexpressing different microRNAs. For each line, unless statedotherwise, two experiments were performed in 50 ml tubes containing 10ml of TAP growth medium and a third experiment was performed in 250 mlflasks containing 50 ml of TAP.

Oil content in transformed lines of C. reinhardtii is depicted in FIGS.2A-2D.

TABLE 6 Oil content in transformed C. Reinhardtii lines overexpressingmicroRNA, relative to oil content in controls. Overexpressed 10 mlmedium 50 ml microRNA line clone Exp 1 Exp 2 medium CR5 in 5 4 1.34 ±0.05 1.34 ± 0.2 1.20 ± 0.04 cre-miR1162 7 1.36 ± 0.16 1.18 ± 0.05 1.25 ±0.11 9 1.71 ± 0.30 0.95 ± 0.09 1.17 ± 0.03 11 1.60 ± 0.17 1.18 ± 0.051.27 ± 0.12 12 1.49 ± 0.27 0.96 ± 0.07 1.18 ± 0.12 18 1.28 ± 0.16 0.94 ±0.1 1.22 ± 0.05 20 1.39 ± 0.18 1.03 ± 0.1 1.26 ± 0.01 28 1.45 ± 0.151.16 ± 0.11 1.55 ± 0.11 miR-1151b 1151 13 1.49 ± 0.09 0.97 ± 0.04 1.09 ±0.02 19 1.45 ± 0.11 0.97 ± 0.07 1.31 ± 0.02 24 1.95 ± 0.84 1.09 ± 0.071.00 ± 0.04 25 1.58 ± 0.1 1.13 ± 0.28 1.76 ± 0.01 30 1.76 ± 0.07 1.09 ±0.11 1.04 ± 0.07 miR-1157 1157 3 2.24 ± 0.84 0.97 ± 0.03 1.08 ± 0.03 121.92 ± 0.41 1.01 ± 0.13 1.17 ± 0.01 13 1.74 ± 0.17 1.01 ± 0.13 1.00 ±0.05 14 1.88 ± 0.21 1.04 ± 0.02 1.12 ± 0.05 16 2.87 ± 0.64 0.99 ± 0.061.14 ± 0.1 22 1.59 ± 0.09 1.18 ± 0.06 1.07 ± 0.03 24 2.21 ± 0.86 0.96 ±0.02 1.24 ± 0.04 miR-121 121 9 NA¹ 1.15 ± 0.33 1.17 ± 0.09 16 NA 1.25 ±0.05 1.41 ± 0.07 19 NA 1.06 ± 0.17 1.24 ± 0.05 20 NA 1.08 ± 0.08 1.13 ±0.06 30 NA 1.11 ± 0.08 1.23 ± 0.08 ¹Not applicable

Example 5 MicroRNA Targets

A differentially expressed microRNA identified, is cre-miR1167 (SEQ IDNO: 21), that was downregulated under nitrate-limiting conditions.cre-miR1167 (SEQ ID NO: 21) is predicted to target a CYP51 homologue,which encodes a cytochrome p450 sterol-14α-demethylase: a conserved keyenzyme in sterol biosynthesis {Lepesheva, 2007 Biochim Biophys Acta.2007 March; 1770(3): 467-477}. We are not aware of any work done incharacterizing CYP51 in algae but cyp51 mutants are considered to belethal in other unicellular organisms. Interestingly, a screen for smallmolecule activators of the integrated stress response (ISR) identifiedtwo related compounds that also activated sterol-regulated genes byblocking cholesterol biosynthesis {Harding, 2005 Cell Metab. 2005December; 2(6): 361-371}. The ISR is known to adapt cells to ER stress,a phenomenon that is modulated by a KDEL receptor like a putative targetof CRMIR136 (SEQ ID NO: 72).

Phosphoglycerate mutase is the target gene of CRMIR152 (SEQ ID NO: 38),which was found to be significantly downregulated under both nitrogenstarvation and strong light (Tables 2b and 3b respectively). This enzymeis involved in glycolysis, which is the process in which glucose isconverted to pyruvate, which can be further converted to acetyl coenzymeA (acetyl CoA). Acetyl CoA is the main substrate for fatty acidbiosynthesis. Accordingly, embodiments of the invention includegeneration of transgenic algal strains which overexpressphosphoglycerate mutase modified such that the binding of CRMIR152 (SEQID NO: 38) is eliminated.

Additionally, the enzyme fructose 1,6 bisphosphatase is the target ofCRMIR5 (SEQ ID NO: 26) which was found to be upregulated under stronglight treatment (Table 3a). This enzyme participates in gluconeogenesis,which is the process in which glucose is re-formed from pyruvate and istherefore the opposite process of glycolysis. Accordingly, embodimentsof the invention further include generating transgenic algal strainswhich overexpress CRMIR5 (SEQ ID NO: 26).

It is possible that CRMIR152 (SEQ ID NO: 38) and CRMIR5 (SEQ ID NO: 26)work together to increase the pool of free acetyl CoA when increased oilbiosynthesis is required by upregulating glycolysis and down regulatinggluconegenesis. Accordingly, the invention further relates to transgenicalgal strains which overexpress both modified phosphoglycerate mutaseand CRMIR5.

Another pair of microRNAs possibly working in co-regulation of theirtargets is CRMIR123 (SEQ ID NO: 36) and CRMIR121 (SEQ ID NO: 35), whichis upregulated in strong light. They are both located on two tandemhairpins in the same locus. CRMIR121 (SEQ ID NO: 35) is predicted totarget a gene encoding histone deacetylase (HDAC) and CRMIR123 (SEQ IDNO: 36) is predicted to target a gene encode histone acetyltransferase(HAT), two enzymes that function as general suppressors or activators oftranscription accordingly. The two microRNAs being physically linkedsuggests they might be under the same regulation and function togetherto keep appropriate balance between HDAC and HAT.

Target prediction can allow manipulation of microRNA regulation byexpressing a microRNA-resistant target; a gene where silent mutationswere introduced in the microRNA-binding site thus bypassing the microRNAregulation. Alternatively, manipulation of microRNA regulation can beperformed by microRNA overexpression. Both these strategies have beenused in plants and have resulted in significant phenotypes alterations.

The Web MicroRNA Designer2 (WMD2) tool {Stephan Ossowski, RebeccaSchwab, Detlef Weigel (2008), The Plant Journal 53 (4), 674-690} wasused to predict the targets of selected microRNAs that were found to bedifferentially expressed relative to changes in lipid content. Thedefault settings of the WMD2 were used to perform the search: up to 5mismatches, hybridization temperature of 23° C. and minimumhybridization energy efficiency of 70% from that of the perfect match(dG).

Some of the genes predicted to be targeted by thedifferentially-expressed microRNAs are known to be stress-regulated orto be involved in stress responses. This includes some of the genespredicted to be targeted by CRMIR136 (SEQ ID NO: 72) such as theJumonji-encoding gene JMJC, which was found to be draught-induced inNicotiana benthamiana and Virus-Induced Gene Silencing (VIGS) of theJMJC gene in N. benthamiana caused relative drought tolerant phenotypes.Another stress-regulated predicted target of CRMIR136 is a KDELreceptor, a protein known to modulate the endoplasmic reticulum (ER)stress response in human cell culture {Yamamoto, 2003 THE JOURNAL OFBIOLOGICAL CHEMISTRY Vol. 278, No. 36, pp. 34525-34532, 2003}. The ERstress response is initiated by an increase in misfolded proteins in theER and includes an induction of the synthesis of ER chaperones,suppression of general protein synthesis and enhancement of theER-associated degradation of misfolded proteins.

5.1 Differential Expression of ADCY33 (SEQ ID NO: 80), a PredictedTarget of CRMIR136 (SEQ ID NO: 72) in Nitrate-limiting and NitrateSufficient Conditions

The adenylate cyclase-encoding gene ADCY33 (SEQ ID NO: 80) is anotherpredicted target of CRMIR136 (SEQ ID NO: 72) and its expression, innitrate-limiting and nitrate sufficient conditions, was assessed byqRT-PCR according as follows:

-   I. cDNa was prepared from the RNA extracted as described in example    3, by the steps of the following protocol:    -   1. 1 μg total RNA in 4 μl DDW.    -   2. Incubate at 85° C. for 2 min and put on ice.    -   3. Add on ice reaction mixture 6 μl:

Component volume (μl) 5X buffer 2 0.1M DTT 1 RNAsin 0.5 Oligo dT primer(10 μM stock) 1 10 mM dNTP's 0.5 RT superscript II 0.5 DDW 0.5

-   -   4. 1 h at 42° c.    -   5. 85° C. for 2 min    -   6. Spin and place on ice.

-   Final concentration: 100 ng/μl, total of 10 μl.

-   Make dilution of 10 ng/μl

-   PCR program 85 42 RT in machine no. 238

-   The sequences used in the PCR procedure are detailed in table 7    below:

TABLE 7 Sequences used in qRT-PCR assay of ADCY33 (SEQ ID NO: 80) expression Role in PCR Sequence SEQ ID NO:Oligo dT primer TTTTTTTTTTTTTTTTTTV 81 Forward primerATGATGGGCCTCATGGTCTA 82 Reverse primer CCAGGATGTCGTTGATGATG 83

-   II. Real time PCR using SYBR mRNA was preformed by the steps of the    following protocol:-   1 Divide the Fwd primers into 96-well plate and spin it down.-   2. Take out the cDNA, defrost and keep on ice.    -   Dilute the cDNA: 100 ng/μl→10 ng/μl    -   Replace tip between adding cDNA and mixing it in the DDW!-   3. In the PCR room assemble (at room temp.) in 2 ml tube:

Component volume SYBR Green 10 μl DDW  7 μl

-   4. Flip the tube, vortex and short spin. Put the mix on ice.-   5. Add (outside of the PCR room, using Gilson 200):

Component volume cDNA 0.05 ng/μl  1 μl Total Vol 18 μl

-   -   Mix by flipping, vortex and short spin.

-   6. Divide 18 μl from the mix into each well, using the stepper from    the PCR room.

-   7. Add 1 μl Fwd primer 1 μl Rev primer.

-   Use the PCR-multi-channel.

-   8. Short spin the plate and start the reaction.

-   9. The reaction program:    Stage 1, Reps=1

-   step 1: Hold @ 95.0 C for 10:00 (MM:SS), Ramp Rate=100    Stage 2, Reps=40

-   Step 1: Hold @ 95.0 C for 0:15 (MM:SS), Ramp Rate=100

-   Step 2: Hold @ 60.0 C for 1:00 (MM:SS), Ramp Rate=100    Dissociation Protocol:

-   Stage 3, Reps=1

-   Step 1: Hold @ 95.0 C for 0:15 (MM:SS), Ramp Rate=Auto

-   Step 2: Hold @ 60.0 C for 1:00 (MM:SS), Ramp Rate=Auto

-   Step 3: Hold @ 95.0 C for 0:15 (MM:SS), Ramp Rate=Auto

-   Standard 7500 Mode

-   Sample Volume (μL): 20.0

-   Data Collection Stage 2, Step 2

As indicated in FIG. 5, the expression of ADCY33 (SEQ ID NO: 80) wasfound by qRT-PCR to be 2.9-fold induced under nitrate-limitingconditions.

Nitrogen starvation is required for inducing sexual differentiation inC. reinhardtii {Goodenough, 2007, Sex determination in Chlamydomonas.Semin Cell Dev Biol 2007, 18(3):350-361}. One of the first eventsfollowing sexual differentiation induction is a protein kinase-dependentactivation of a flagellar adenylate cyclase that causes a 10 foldincrease in cAMP concentrations {Zhang et al., 1996; Celladhesion-dependent inactivation of a soluble protein kinase duringfertilization in Chlamydomonas. Mol Biol Cell 1996, 7(4):515-527}. Thefinding that the expression of ADCY33 (SEQ ID NO: 80) is upregulatedunder nitrate-limiting conditions correlates with the need of increasedcAMP levels during C. reinhardtii sexual differentiation.

5.2 Introduction of microRNA Resistant Target Genes

Synthetic DNA clones containing the cDNA sequences of the miR-resistanttargets according to table 8 below were synthesized by Genscript.

TABLE 8 Sequences of microRNA resistant target genes microRNA resistantmiR target gene SEQ ID NO CRMIR152 (SEQ ID NO: 38) 86 CR167 (SEQ ID NO:84) 87 cre-miR1167 (SEQ ID NO: 21) 88The clones were designed to contain an NdeI site at their 5′ end and anEcoRI site at their 3′ end and were constructed in the pUC19 vector. Thevectors were digested with NdeI and EcoRI to release the fragmentscontaining the indicated sequences, which were cloned to the pGenD-Blevector (N. Fischer & J. D. Rochaix, Molecular General Genomics, 2001,265:888-894). pGenD-Ble was previously digested with NdeI and EcoRI torelease the ble gene. The digested pGenD-Ble vector was separated by gelelectrophoresis and the fragment containing the vector minus the blegene was excited from the gel.

The resulting vector containing the pGenD promoter-miR-resistanttarget-and pGenD terminator were designated pGenD-CR152R, pGenD-167R andpGenD-1167R. The vectors were co-transformed into Chlamydomonasreinhardtii cw15 together with pGenD-Aph following the method describedby K Kindle (K. L. Kindle, Proc. Natl. Acad. Sci. U.S.A. 93 (1990)13689-13693). PgenD-Aph was prepared by introducing the Aph gene (I.Sizova, M. Fuhrmann and P. Hegemann, Gene 277 (2001) 221-229) betweenthe NdeI and EcoRI sites of pGenD-Ble as described above. Cells wereselected on TAP medium containing 15 microgram/ml paromomycin.

The foregoing description of the specific embodiments so fully revealsthe general nature of the invention that others can, by applying currentknowledge, readily modify and/or adapt for various applications suchspecific embodiments without undue experimentation and without departingfrom the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. Althoughthe invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

It should be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

What is claimed is:
 1. A transgenic plant comprising an expressionconstruct comprising a heterologous promoter operably linked to anucleic acid molecule having at least 95% identity to SEQ ID NO:
 38. 2.The transgenic plant of claim 1, selected from the group consisting ofCorn, Rapeseed/Canola, Soybean, Sugarcane, Sugar Beet, Barley, Wheat,Jatropha, Castor Bean, Chinese Tallow, Palm, Camelina, and Mustard. 3.The transgenic plant of claim 1, wherein the plant is algae.
 4. Thetransgenic plant of claim 3, wherein said algae is selected from thegroup consisting of Neochloris oleoabundans, Scenedesmus dimorphus,Euglena gracilis, Phaeodactylum tricornutum, Pleurochrysis carterae,Tetraselmis chui, Tetraselmis suecica, Isochrysis galbana,Nannochloropsis salina, Nannochloropsis oculata, Botryococcus braunii,Dunaliella tertiolecta, Nannochloris sp., Spirulina sp., Chlorella sp.,Crypthecodinium cohnii, Cylindrotheca sp., Dunaliella primolecta,Monallanthus salina, Nitzschia sp., Schizochytrium sp. and Tetraselmissueica.
 5. The transgenic plant of claim 3, wherein said algae isselected from the group consisting of Chlamydomonas reinhardtii,Chlorella vulgaris, and Phaeodactylum tricornutum.
 6. A vectorcomprising a nucleic acid sequence, wherein said nucleic acid sequencecomprises a heterologous promoter operably linked to a nucleic acidmolecule having at least 95% identity to SEQ ID NO:
 38. 7. The vector ofclaim 6, wherein said heterologous promoter is selected from the groupconsisting of a constitutive promoter, an inducible promoter, and atissue-specific promoter.
 8. A seed obtained from a transgenic plant ofclaim 1, wherein said seed comprises said expression construct.
 9. Aseed obtained by use of the vector of claim 6, wherein said seedcomprises said vector.